1. Field of the Invention
There is substantial interest in employing industrial unicellular microorganism strains as hosts with recombinant DNA to produce polypeptides in high yields. Many industrially important enzymes, such as amylolytic and proteolytic enzymes, are produced by microorganisms of the genus Bacillus, e.g., B. subtilis, B. amylolique-faciens, B. licheniformis, B. stearothermophilus and B. coagulans. In fermenters, strains are employed which are highly robust and stable. Furthermore, the strains are resistant to phage infection and, in addition, to genetic exchange, that is introduction of DNA by conventional transformation procedures. The conventional industrial strains are also prototrophic, in order to avoid adding expensive amino acids to the nutrient medium. Other characteristics of industrial strains are their high productivity until the end of the fermentation, which can be as long as a week, stable cell concentration upon exhaustion of the broth, and high productivity, usually at least 0.5% w/v of a specific secreted protein. In addition, it is often found with Bacilli, that there is a substantial secretion of DNAses, so that there is substantial degradation of any DNA in the medium.
Due to-the genetic modification resistant nature of the industrial strains and their prototrophic character which makes them difficult to starve, they show resistance to transformation. It would therefore be of great value to provide for an efficient process for introducing DNA into industrial strains, where the DNA would be stably maintained in the industrial strain, there would be no loss or substantially no loss of viability and activity of the industrial strain and high yields of endogenous and exogenous polypeptide or protein products could be obtained.
Furthermore, selection of cells is difficult where the modification or transformation of the host cells involves increasing the copy number of an endogenous gene or previously introduced gene, where the gene is not involved with survival selection. Stability of extra copies of an endogenous gene or new genes is of utmost importance to ensure possible use of such genetically modified strains for industrial purposes.
It is therefore highly desirable to have an efficient process in which the stable presence of additional genes (increased copy number) can be detected for and stable chromosomal integration of these additional genes can be selected for.
2. Description of the Prior Art
Genetic manipulations of B. subtilis have been reported by Yoneda et al., Biochem. Biophys. Res. Commun. (1973) 50:765-770; Yoneda and Maruo, J. Bacteriol. (1975) 124:48-54; Sekiguchi et al., J. Bacteriol. (1975) 121:688-694; Hitotsuyanagi et al., Agri. Biol. Chem. (1979) 43:2342-2349; Yoneda, Appl. Env. Microbiol. (1980) 39:274-276.
Introduction of DNA into Bacillus without using conventional procedures for transformation of competent cells has been described. Protoplast transformation for several species of gram positive microorganisms is, for example, reported. Protoplast transformation of Bacillus subtilis has been described by Chang and Cohen. Molec. Gen. Genet. (1979) 168:111-115. Similar protocols were described: for the transformation of Bacillus megaterium protoplasts by Vorobjeva et al., FEMS Microbiol. Lett. (1980) 7:261-263 and by Brown and Carlton, J. Bacteriol. (1980) 142:508-512; the transformation of Bacillus amylolique-faciens protoplasts by Smith et al., Applied Environmental Microbiol. (1986) 51:634-639; for the transformation of Bacillus thuringiensis protoplasts by Fisher, Lithy and Schweitzer, Arch. Microbiol. (1984) 139:213-217 and by Miteva et al., FEMS Microbiol. Lett. (1981) 12:253-256; for Bacillus sphaericus by McDonald and Burke, J. Gen. Microbiol. (1984) 130:203-208; for Clostridium acetobutyliticum by Lin and Blascheck, Appl. Environ. Microbiol. (1984) 48:737-742; for Streptococcus lactis by Kondo and McKay, Appl. Environ. Microbiol. (1984) 48:252-259.
Fusion of bacterial protoplasts has been reviewed by Hopwood, Ann. Rev. Microbiol. (1981) 35:237-272, and this technique has been shown to be useful in the transfer of plasmids among different species by Dancer, J. Gen. Microbiol. (1980) 121:263-266.
Apart from polyethylene glycol induced protoplast fusion and protoplast transformation, other methods of introducing DNA into protoplasts can be developed such as fusion with DNA containing liposomes, as described by Holubova et al., Folia Microbiol. (1985) 30:97-100.
Successful applications of recombinant DNA technology with respect to production improvements of certain, efficiently transformable laboratory strains of B. subtilis have been reported, e.g., .alpha.-amylases, .beta.-lactamases, dihydrofolate reductase, interferon and insulin (Palva, Gene (1982) 19:81-87; Shinomiya et al., Agric. Biol. Chem. (1981) 45:1733-1735; Gray and Chang, J. Bacteriol. (1981) 145:422-428; Williams et al., Gene (1981) 16:199-206; Palva, Gene (1983) 22:229-235). The difficulties in genetically manipulating Bacillus licheniormis soil isolates is reported by Thorne and Stull, J. Bacteriol. (1966) 91:1012-1014 and by Maruo and Tojo, J. Gen. Appl. Microbiol. (1985) 31:323-328. Segregational and structural instability of recombinant plasmids is considered as a major problem in using Bacillus, more specifically Bacillus subtilis as a host organism. These problems have been described by Ehrlich and Niaudet and by Kreft and Hughes in Current Top. Microbiol. Immunol. (1982) 96:19-29 and 1-17, respectively (ed. Holscheider and Goebel, Springer Verlag, N.Y.).
Chromosomal integration and chromosomal amplification of genes in prokaryotic genomes has been reported by Gutterson and Koshland, Proc. Natl. Acad. Sci. USA (1983) 80:4894-4898; Saito, Anzai and Kawamura, Genetics of Industrial Microorganisms, Proc. of the 11th Int. Symposium, ed. Ikeda and Beppu, Tokyo (1982) 125-130; Young, Gen. Microbiol. (1984) 130:1613-1621, Williams and Szalay, Gene (1983) 24:37-51 and in PCT Patent Application 84/00381. See also, U.K. Patent Application 2091628; European Patent Application 0 034 470; European Patent Application 0 032 238; and European Patent Application 0 077 109, which disclosure is incorporated herein by reference, as it relates to pUR1523.